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Abstract:

The present invention relates to a novel monascuspurpurone compound of
formula (I):
##STR00001##
or a pharmaceutically acceptable derivative thereof as described in the
specification, the process for preparation of the same, and the
composition comprising the same. The uses to of a monascuspurpurone
compound for promoting adipocyte differentiation, for increasing the
activity of PPARγ and/or C/EBPα, for lowering blood glucose,
for preventing and/or treating a disease or disorder related to insulin
resistance, and for preventing and/or treating metabolic syndrome or its
complications are also provided.

Claims:

1. A method for promoting adipocyte differentiation in a subject, which
comprises administering to said subject an effective amount of the
compound of formula (I) or a pharmaceutically acceptable derivative
thereof, wherein the compound of formula (I) is ##STR00006## wherein
R1 and R2 are independently selected from the group consisting
of alkyl, alkenyl, and carbonyl and R3 is alkyl.

2. A method of preventing and/or treating a disease or disorder related
to insulin resistance in a subject, which comprises administering to said
subject an effective amount of the compound of formula (I) or a
pharmaceutically acceptable derivative thereof of claim 1.

3. A method for increasing the activity of PPARγ and/or
C/EBPα in a subject in need thereof, which comprises administering
to said subject an effective amount of the compound of formula (I) or a
pharmaceutically acceptable derivative thereof of claim 1.

4. A method of lowering blood glucose in a subject, which comprises
administering to said subject an effective amount of the compound of
formula (I) or a pharmaceutically acceptable derivative thereof of claim
1.

5. A method of preventing and/or treating metabolic syndrome or its
complication in a subject, which comprises administering to said subject
an effective amount of the compound of formula (I) or a pharmaceutically
acceptable derivative thereof of claim 1.

6. The method of claim 5, wherein the metabolic syndrome or its
complication is selected from the group consisting of abdominal obesity,
atherogenic dyslipidemia, elevated blood pressure, insulin resistance or
glucose intolerance, type 2 diabetes and cardiovascular disease.

7. The method of claim 6, wherein a second therapeutic agent for
preventing or treating metabolic syndrome or its complication is
administered to the subject.

Description:

[0001] This application is a Divisional of U.S. application Ser. No.
13/336,234, filed on Dec. 23, 2011, which claims priority to U.S.
provisional application Ser. No. 61/426,801, filed on Dec. 23, 2010.

FIELD OF THE INVENTION

[0002] This invention relates to novel monascuspurpurones and the process
for preparation of the same. It further relates to the compositions
comprising a monascuspurpurone compound, and the uses of a
monascuspurpurone compound for promoting adipocyte differentiation, for
increasing the activity of PPARγand/or C/EBPα, for lowering
blood glucose, and for preventing and/or treating a disease or disorder
related to insulin resistance, such as metabolic syndrome. The invention
also relates to the use of extracts of red yeast-fermented products for
preventing and/or treating a disease or disorder related to insulin
resistance, such as metabolic syndrome.

[0005] 231-236) found that LDL, total cholesterol, and triglycerides in
subjects suffering from hyperlipidemia decreased by 22%, 17%, and 11%,
respectively, in the experimental group treated with the red yeast rice
fermented with Monascus spp., while those in the control group maintained
the original values. The study also confirmed that red yeast rice
fermented with Monascus spp. is superior to conventional lipid-lowering
drugs because it does not adversely affect liver function or cause other
side effects. According to a report by the Shanghai First People's
Hospital regarding the effect of red yeast rice fermented with Monascus
spp. as compared with Pravastatin in treating hyperlipidemia (see Yang
HT., et al., 1997, "A comparative Study of Xuezhikang and Mevalotin in
treatment of Essential Hyperlipidemia." Chinese Journal of New Drugs, 6:
265-268), total cholesterol, low density lipoprotein cholesterol and Apo
B decreased by 26.59% vs 18.92%, 33.32% vs 24.24%, and 18.42% vs 8.89%,
respectively, showing that the red yeast rice fermented with Monascus
spp. has a significantly superior effect. A study conducted at the
Chinese Academy of Medical Sciences Deng Sisuo Hospital (see Lin, Feng.,
1992, "Evolution of research and development of Monascus." Scientific
agriculture, 40: 193-198) also confirmed that red yeast rice fermented
with Monascus spp. is effective in treatment of subjects with high
cholesterol. Red yeast rice fermented with Monascus spp. has since become
commercialized as a lipid-lowering drug and for prevention of coronary
heart disease.

[0007] Modulating lipid metabolism is one strategy for the treatment of
metabolic syndrome. Thiazolidinediones (TZDs) are type 2 diabetes drugs
developed in the early 1980s. Studies on the mechanisms of TZDs showed
that they increase insulin sensitivity by activating PPARγ. One of
the characteristic effects of activating PPARγ is increasing
differentiation of adipocytes. Increasing adipocyte differentiation has
therefore become a popular method for screening agents that have
potential in activating PPARγ and decreasing insulin resistance.

[0008] However, the prior arts do not disclose which compounds in the red
yeast rice fermented with Monascus spp. have the anti-diabetic effects
and what their pharmacological mechanism is.

SUMMARY OF THE INVENTION

[0009] One purpose of the invention is to provide a compound of formula
(I):

##STR00002##

[0010] or a pharmaceutically acceptable derivative thereof,

[0011] wherein R1 and R2 are independently selected from the
group consisting of alkyl, alkenyl, and carbonyl, and R3 is alkyl.

[0012] Another purpose of the present invention is to provide a
composition comprising the compound of formula (I) or a pharmaceutically
acceptable derivative thereof, and optionally a pharmaceutically
acceptable carrier or excipient.

[0013] Another purpose of the present invention is to provide a process
for the preparation of the compound of formula (I) or a pharmaceutically
acceptable derivative thereof.

[0014] Another purpose of the present invention is to provide a red yeast
rice extract.

[0015] Another purpose of the present invention is to provide a
composition comprising the red yeast rice extract of the invention, and
optionally a pharmaceutically acceptable carrier or excipient.

[0016] Another purpose of the present invention is to provide a method for
promoting adipocyte differentiation in a subject.

[0017] Another purpose of the present invention is to provide a method for
increasing the activity of PPARγ and/or C/EBPα.

[0018] Another purpose of the present invention is to provide a method of
preventing and/or treating a disease or disorder related to insulin
resistance in a subject.

[0019] Another purpose of the present invention is to provide a method of
lowering blood glucose in a subject.

[0020] Still another purpose of the present invention is to provide a
method of preventing and/or treating metabolic syndrome or its
complications.

[0021] The present invention is described in detail in the following
sections. Other characteristics, purposes and advantages of the present
invention can be easily found in the detailed description and claims.

[0025] FIGS. 4(A) to 4(D) show the effects of insulin (A) and Compound 1
(in the concentrations of 2 μg/ml (B), 5 μg/ml (C) and 10 μg/ml
(D)) on the differentiation of 3T3-L1 preadipocytes.

[0026] FIG. 5 shows the real-time PCR results of the adipocyte
differentiation related gene expressions of the adipocytes treated with
Compound 1 on day 4.

[0027] FIG. 6 shows the real-time PCR results of the adipocyte
differentiation related gene expressions of the adipocytes treated with
Compound 1 on day 7.

[0028] FIG. 7 shows the real-time PCR results of the adipocyte
differentiation related gene expressions of the adipocytes treated with
Compound 1 on day 9.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention can be more readily understood by reference
to the following detailed description of various embodiments of the
invention, the examples, and the chemical drawings and tables with their
relevant descriptions. It is to be understood that unless otherwise
specifically indicated by the claims, the invention is not limited to
specific preparation methods, carriers or formulations, or to particular
modes of formulating the compounds of the invention into products or
compositions intended for topical, oral or parenteral administration,
because as one of ordinary skill in the relevant arts is well aware, such
things can, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only and is not intended to be limiting.

Definitions

[0030] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to have
the following meaning:

[0031] As used herein, the term "alkyl" and "alkenyl" include straight and
branched chains.

[0032] "Alkyl" refers to a hydrocarbon group that can be conceptually
formed from an alkane by removing hydrogen from the structure of a
non-cyclic hydrocarbon compound having straight or branched carbon
chains, and replacing the hydrogen atom with another atom or organic or
inorganic substituent group. In some embodiments of the invention, the
alkyl groups are "C1 to C10 alkyl" such as methyl, ethyl,
propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, amyl,
tert-amyl, hexyl and the like. Many embodiments of the invention comprise
"C1 to C7 alkyl" groups that include methyl, ethyl, propyl,
iso-propyl n-butyl, iso-butyl, sec-butyl, t-butyl, pentyl, hexyl, and
heptyl groups.

[0033] The term "alkenyl" is structurally analogous to an alkyl group or
residue that comprises at least one carbon-carbon double bond. In some
embodiments, the alkenyl groups are "C2 to C7 alkenyls" which
are exemplified by vinyl, allyl, propenyl, 2-butenyl, 3-butenyl,
2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,
5-hexenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, and
6-heptenyl, as well as dienes and trienes of straight and branched
chains. In other embodiments, alkenyls are limited to two to four carbon
atoms.

[0034] As used herein, the term "carbonyl" includes straight and branched
chains.

[0035] "Carbonyl" refers to a functional group comprising a carbon atom
double-bonded to an oxygen atom: C═O. In some embodiments of the
invention, the carbonyl group is "C1 to C10 carbonyl."

[0036] The term "a pharmaceutically acceptable derivative" or
"pharmaceutically acceptable derivatives" as used herein denotes a
compound that is modified from the compound of the invention but that has
properties and efficacies that are the same as or better than those of
the compound of the invention. Preferably, the pharmaceutically
acceptable derivative is a pharmaceutically acceptable salt, solvate,
hydrate, or prodrug of the compound of the invention.

[0037] One or more of the compounds of the invention may be present as a
salt. The term "salt" encompasses those salts formed with the organic and
inorganic anions and cations. Furthermore, the term includes salts that
form by standard acid-base reactions with basic groups and organic or
inorganic acids. Such acids include hydrochloric, hydrofluoric,
trifluoroacetic, sulfuric, phosphoric, acetic, succinic, citric, lactic,
maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic,
D-camphoric, glutaric, phthalic, tartaric, lauric, stearic, salicyclic,
methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, and cinnamic
acids.

[0038] The compounds of the invention can also exist as solvates and
hydrates. Thus, these compounds may crystallize with, for example, waters
of hydration, or one, a number of, or any fraction of molecules of the
mother liquor solvent. The solvates and hydrates of such compounds are
included within the scope of this invention.

[0039] The term "subject" as used herein denotes any animal, preferably a
mammal, and more preferably a human. Examples of subjects include humans,
non-human primates, rodents, guinea pigs, rabbits, sheep, pigs, goats,
cows, horses, dogs and cats.

[0040] The term "effective amount" of a compound as provided herein means
a sufficient amount of the compound to provide the desired regulation of
a desired function, such as gene expression, protein function, or the
induction of a particular type of response. As will be pointed out below,
the exact amount required will vary from subject to subject, depending on
the disease state, physical conditions, age, sex, species and weight of
the subject, the specific identity and formulation of the composition,
etc. Dosage regimens may be adjusted to induce the optimum therapeutic
response. For example, several divided doses may be administered daily or
the dose may be proportionally reduced as indicated by the exigencies of
the therapeutic situation. Thus, it is not possible to specify an exact
"effective amount." However, an appropriate effective amount can be
determined by one of ordinary skill in the art using only routine
experimentation.

[0041] The term "preventing" or "prevention" is recognized in the art, and
when used in relation to a condition, it includes administering, prior to
onset of the condition, an agent to reduce the frequency or severity of
or delay the onset of symptoms of a medical condition in a subject
relative to a subject which does not receive the agent.

[0042] The term "treating" or "treatment" as used herein denotes
reversing, alleviating, inhibiting the progress of, or improving the
disorder or condition to which such term applies, or one or more symptoms
of such disorder or condition.

[0043] The term "carrier" or "excipient" as used herein refers to any
substance, not itself a therapeutic agent, used as a carrier and/or
diluent and/or adjuvant, or vehicle for delivery of a therapeutic agent
to a subject or added to a formulation to improve its handling or storage
properties or to permit or facilitate formation of a dose unit of the
composition into a discrete article such as a capsule or tablet suitable
for oral administration. Suitable carriers or excipients are well known
to persons of ordinary skill in the art of manufacturing pharmaceutical
formulations or food products. Carriers or excipients can include, by way
of illustration and not limitation, buffers, diluents, disintegrants,
binding agents, adhesives, wetting agents, polymers, lubricants,
glidants, substances added to mask or counteract a disagreeable taste or
odor, flavors, dyes, fragrances, and substances added to improve
appearance of the composition. Acceptable carriers or excipients include
citrate buffer, phosphate buffer, acetate buffer, bicarbonate buffer,
stearic acid, magnesium stearate, magnesium oxide, sodium and calcium
salts of phosphoric and sulfuric acids, magnesium carbonate, talc,
gelatin, acacia gum, sodium alginate, pectin, dextrin, mannitol,
sorbitol, lactose, sucrose, starches, gelatin, cellulosic materials (such
as cellulose esters of alkanoic acids and cellulose alkyl esters), low
melting wax cocoa butter, amino acids, urea, alcohols, ascorbic acid,
phospholipids, proteins (for example, serum albumin), ethylenediamine
tetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), sodium chloride or
other salts, liposomes, mannitol, sorbitol, glycerol or powder, polymers
(such as polyvinyl-pyrrolidone, polyvinyl alcohol, and polyethylene
glycols), and other pharmaceutically acceptable materials. The carrier
should not destroy the pharmacological activity of the therapeutic agent
and should be non-toxic when administered in doses sufficient to deliver
a therapeutic amount of the agent.

[0044] Often, ranges are expressed herein as from "about" one particular
value and/or to "about" another particular value. When such a range is
expressed, an embodiment includes the range from the one particular value
and/or to the other particular value. Similarly, when values are
expressed as approximations, by use of the word "about," it will be
understood that the particular value forms another embodiment. It will be
further understood that the endpoints of each of the ranges are
significant both in relation to and independently of the other endpoint.

[0045] "Optional" or "optionally" means that the subsequently described
event or circumstance may or may not occur, and that the description
includes instances where said event or circumstance occurs and instances
where it does not. For example, the phrase "optionally comprise an agent"
means that the agent may or may not exist.

[0046] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include plural
referents unless the context clearly dictates otherwise. Thus, unless
otherwise required by context, singular terms shall include the plural
and plural terms shall include the singular.

The Compounds of the Invention

[0047] The present invention relates to a prupurone or a pharmaceutically
acceptable derivative thereof. The monascuspurpurone of the invention has
the following formula (I):

##STR00003##

[0048] wherein R1 and R2 are independently selected from the
group consisting of alkyl, alkenyl, and carbonyl, and R3 is alkyl.

[0049] In some preferred embodiments of the compound of formula (I),
R1 and R2 are independently C1-C10 alkyl,
C2-C7 alkenyl or C1-C10 carbonyl. More preferably,
R1 is hexanoyl. In another aspect, R2 is more preferably
2-oxopentyl.

[0050] In some preferred embodiments of the compound of formula (I),
R3 is C1-C10 alkyl. More preferably, R3 is methyl.

[0051] In a most preferred embodiment, the compound of formula (I) is
rel-(1S,7aR,3aR)-1-hexanoyl-5,13-dimethyl-6-(2-oxopentyl)-7a,7-dihydroben-
zofuran-2,4-dione.

[0052] The compounds of the invention are preferably further converted
into a pharmaceutically acceptable derivative, such as a pharmaceutically
acceptable salt, solvate or prodrug, by any known methods.

The Compositions of the Invention

[0053] The present invention also provides a composition comprising the
compound of the invention or a pharmaceutically acceptable derivative
thereof. The composition of the invention is preferably a food
composition or a pharmaceutical composition. The compound of formula (I)
of the present invention in the composition is preferably provided in the
form of an extract of red yeast rice or a chemical compound.

[0054] The pharmaceutical composition of the invention is preferably
administered topically or systemically by any method known in the art,
including, but not limited to, intramuscular, intradermal, intravenous,
subcutaneous, intraperitoneal, intranasal, oral, mucosal or external
routes. The appropriate route, formulation and administration schedule
can be determined by those skilled in the art. In the present invention,
the pharmaceutical composition can be formulated in various ways,
according to the corresponding route of administration, such as a liquid
solution, a suspension, an emulsion, a syrup, a tablet, a pill, a
capsule, a sustained release formulation, a powder, a granule, an
ampoule, an injection, an infusion, a kit, an ointment, a lotion, a
liniment, a cream or a combination thereof. If necessary, it may be
sterilized or mixed with any pharmaceutically acceptable carrier or
excipient, many of which are known to one of ordinary skill in the art;
see paragraph

[0037] for example.

The Preparation Processes of the Invention

[0055] The present invention provides a process for the preparation of the
compound of formula (I):

##STR00004##

[0056] or a pharmaceutically acceptable derivative thereof,

[0057] wherein R1 and R2 are independently selected from the
group consisting of alkyl, alkenyl, and carbonyl, and R3 is alkyl.

[0058] In one preferred embodiment, the process of the invention comprises
the steps of:

[0064] According to the process of the invention, the isolated strain can
be Monascus pilosus, Monascus purpureus, or Monascus ruber, preferably
Monascus purpureus; more preferably Monascus purpureus M615 (DSM 24162).

[0065] According to the process of the invention, prior to step (b), the
red yeast rice is preferably dried.

[0066] According to the process of the invention, step (a) preferably
comprises fermenting rice with an isolated strain of Monascus spp. in the
presence of tartaric acid to obtain red yeast rice.

[0067] According to the process of the invention, the ratio of ethyl
acetate and H2O in step (c) is preferably about 1:1.

[0068] According to the process of the invention, step (d) preferably
comprises loading the ethyl acetate-soluble fraction into a
chromatographic column with silica gel and eluting the column with an
eluent comprising n-hexane/ethyl acetate 10:1 to 5:1 to produce fifteen
fractions. The eluted fraction used in step (e) is Fraction 10.

[0069] According to a preferred process of the invention, step (e) uses
TLC with n-hexane/EtOAc, 5:1 as solvent.

[0070] The present invention provides processes for the preparation of red
yeast rice extracts. In one preferred embodiment, the process comprises
the steps of:

[0074] According to the red yeast rice extract preparation process of the
invention, prior to step (b), the red yeast rice is preferably dried.

Utilities

[0075] One aspect of the therapeutic method of the present invention is to
promote adipocyte differentiation in a needed subject, which comprises
administering to said subject an effective amount of a compound of
formula (I)

##STR00005##

[0076] or a pharmaceutically acceptable derivative thereof,

[0077] wherein R1 and R2 are independently selected from the
group consisting of alkyl, alkenyl, and carbonyl, and R3 is alkyl;
or a red yeast rice extract.

[0078] Another aspect of the therapeutic method of the present invention
is to increase the activity of PPARγ and/or C/EBPα.
Preferably, the compound of formula (I) or a pharmaceutically acceptable
derivative thereof, or a red yeast rice extract promotes adipocyte
differentiation through the PPARγ/C/EBPα pathway.

[0079] Another aspect of the therapeutic method of the present invention
is to lower blood to glucose in a subject, which comprises administering
to said subject an effective amount of the compound of formula (I) or a
pharmaceutically acceptable derivative thereof, or a red yeast rice
extract.

[0080] Another aspect of the therapeutic method of the present invention
is to prevent and/or treat a disease or disorder related to insulin
resistance in a subject, which comprises administering to said subject an
effective amount of the compound of formula (I) or a pharmaceutically
acceptable derivative thereof, or a red yeast rice extract.

[0081] In certain embodiments, the disease or disorder related to insulin
resistance is metabolic syndrome or its complications, such as
atherogenic dyslipidemia, elevated blood pressure, insulin resistance or
glucose intolerance, type 2 diabetes or cardiovascular disease.

[0082] According to the methods of the present invention, the compound of
formula (I) or a pharmaceutically acceptable derivative thereof is
preferably administered topically or systemically by any method known in
the art, including, but not limited to, intramuscular, intradermal,
intravenous, subcutaneous, intraperitoneal, intranasal, oral, mucosal or
external routes. The appropriate route, formulation and administration
schedule is determined by those skilled in the art.

[0083] According to the methods of the present invention, the compounds of
formula (I) or a pharmaceutically acceptable derivative thereof is
preferably administered in combination with a second agent effective in
preventing and/or treating metabolic syndrome or its complications,
thereby improving the therapeutic effect of the compounds of the
invention. Many agents are known in the art to be effective in preventing
and/or treating metabolic syndrome or its complications. Examples of such
agents include, but are not limited to, drugs to control cholesterol
levels and lipids, such as statins, fibrates, or nicotinic acid; drugs to
control high blood pressure, such as diuretics or angiotensin-converting
enzyme (ACE) inhibitors; and drugs to control high blood sugar, such as
metformin, insulin, sulfonylurea to (SU), biguanide, α-glucosidase
inhibitors, thiazolidinediones (TZDs) and the like.

[0084] The following examples are provided to aid those skilled in the art
in practicing the present invention.

EXAMPLES

Microorganism

[0085] Monascus purpureus M615 was deposited with the Deutsche Sammlung
von Mikroorganismen and Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b,
D38124, Braunschweig, Germany, DSMZ on 28 Oct. 2010 in accordance with
the Budapest Treaty and assigned accession number DSM 24162. It was also
deposited with the Bioresource Collection and Research Center (BCRC) of
the Food Industry Research and Development Institute (FIRDI), 331
Shih-Pin Road, 300, Hsinchu, Taiwan, R.O.C., on 27 Oct. 2010 and assigned
accession number BCRC 930146.

Example 1

Preparation of Yeast Material

[0086] M. purpureus M615 was inoculated on a Potato Dextrose Agar (PDA)
(Difco, USA) plate and incubated at 30° C. for 7 days. The spores
were washed out from the PDA plate using 6 ml of sterile water and 1 ml
of the spore suspension was inoculated in a 250 ml Erlenmeyer flask
containing GSP medium (which contains 7% of glycerol, 3% of flour, 1.2%
of polypeptone, 3% of soybean powder, 0.1% of magnesium sulfate and 0.2%
of sodium nitrate) and shaken and incubated at 30° C., 150 rpm for
three days to obtain the yeast material stock.

Example 2

Solid Fermentation

[0087] 75 ml of 0.2% tartaric acid solution was added to each of ten
450-ml wide-mouth glass bottles each containing 75 g of Zailai rice (long
grain rice). The rice was soaked at 4° C. overnight. Then, the
liquid was drained off and the rice was sterilized. An aliquot of 7.5 ml
of the yeast material stock obtained as mentioned above was inoculated in
each bottle and incubated at 25° C. for 14 days (at the 7th day of
the incubation, 7.5 ml of GSP medium were added to the culture) to obtain
red yeast rice.

[0094] Twenty-one C-atom signals (Table 1) corresponding to seven
quaternary C-atoms, two CH, eight CH2, and four Me groups were
observed in the 13C-NMR and DEPT spectra. The 1H-NMR spectrum
of 1 exhibited signals attributed to one allylic Me (δ(H) 1.79 (br.
t, J=1.8 Hz, Me-C(5))), signals of α-methylene protons of two
ketones (δ(H) 2.47 (t, J=7.6 Hz, CH2(15))), 2.57 (t, J=7.6 Hz,
CH2(10))), 3.25 (d, J=17.0 Hz, 1H of CH2(8)), and 3.65 (d,
J=17.0 Hz, 1H of CH2(8))), four β-methylene protons of two
ketones (6(H) 1.57-1.67 (m, CH2(11) and CH2(16))), one
methylene protons (δ(H) 2.58 (dd, J=18.0, 4.4 Hz, 1H of
CH2(7)), and 2.99 (dd, J=18.0, 10.8 Hz, 1H of CH2(7))), and two
terminal Me moieties (δ(H) 0.87 (t, J=7.6 Hz, Me(19)) and 0.94 (t,
J=7.6 Hz, Me(12)). The 13C-NMR and DEPT spectra exhibited the
presence of four C═O carbonyl functions including one
α,β-unsaturated C═O group (δ(C) 193.4 (C(4)), one
lactone C═O group (6(C) 169.5 (C(2)), and two saturated ketone groups
(δ(C) 202.4 (C(14)) and 205.9 (C(9)), one C═C bond (δ(C)
131.9 (C(6)) and 148.2 (C(5))), and one oxymethyl group (6(C) 17.2
(C(13))). Since five out of seven unsaturation equivalents were accounted
for by the above-mentioned 13C-NMR data, 1 was inferred to have two
rings (ring A as a six-membered and ring B as a five-membered ring). In
addition, rings A and B were further determined as a cyclohex-2-enone
backbone combined with one γ-lactone ring by the following HMBC and
COSY analyses.

[0096] Further confirmation by 1H-1H COSY plot (H--C(1)
(δ(H) 3.64)/H--C(7a) (δ(H) 3.16)/CH2(7) (δ(H)
2.58/2.99)) and by the HMBC correlations of Me(13) and C(3a), C(7a), and
C(4); H--C(7a)/C(3a) and C(2); CH2(7)/C(3a) and C(7a); Me(5)/C(4),
C(5), and C(6) determined the skeleton of Compound 1 to be
5,13-dimethyl-7a,7-dihydrobenzofuran-2,4-dione (rings A & B).

[0097] HMBC correlations between the H-atom signal at δ(H) 3.25/3.65
(CH2(8)) and the C-atom signals at δ(C) 131.9 (C(6)), and
148.2 (C(5)) established the position of the 2-oxopentyl group at C(6).
The HMBC showed correlations between the H-atom at δ(H) 2.47
(CH2(15)) and the C-atom at δ(C) 54.8 (C(1)); and between the
H-atom at δ(H) 3.16 (H--C(7a)) and the C-atom at δ(C) 202.4
(C(14)) can verify the connection of the hexanoyl group to the
γ-lactone ring at C(1). The other key correlations of HMBC are
illustrated in FIG. 1.

[0098] The relative configuration of Compound 1 was derived by a NOESY
spectrum (FIG. 2) in combination with biogenetic considerations and
comparison with similar compound. NOEs for Me-C(13)/H--C(1),
Me-C(13)/Hax--C(7), and H--C(1)/Hax--C(7) indicated that
Me-C(13), H--C(1), and Hax--C(7) were on the same side of the
molecular plane, tentatively assumed as β-orientation. The H--C(7a)
was α-oriented which was further confirmed by the NOE
H--C(7a)/Heq--C(7). Thus, the trans-configuration for H--C(7a)
(δ(H) 3.16)/H--C(1) (δ(H) 3.64) was deduced from no NOE
correlation between H--C(7a) and H--C(1) and coupling constant 3J
value 12.8 Hz between H--C(1)/H--C(7a) and H--C(7a)/Hax--C(7). The
H--C(7a)/CH2(15) and CH2(7)/CH2(8) NOESY cross-peaks
report on a preferred conformation of the hexanoyl and 2-oxopentyl groups
around the C(1)-C(14) and C(6)-C(8) bonds, respectively. On the other
hand, the NOE cross peaks CH2(8)/Me-C(5), CH2(10)/CH2(11),
and CH2(10)/Me-C(12) were also observed in FIG. 2. Based on the
information from the 1H NMR, COSY, and NOESY spectra, a
computer-generated 3D structure was obtained by using the abovementioned
molecular modeling program with MM2 force-field calculations for energy
minimization (FIG. 3). The calculated distances between H--C(1)/Me-C(13)
(2.396 Å) are less than 4.00 Å, and between H--C(1)/H-C(7a)
(4.486 Å) and Me-C(13)/H--C(7a) (4.320 Å) are more than 4.00
Å; this is consistent with the well-defined NOESY (FIG. 2) observed
for each of the proton pairs. Consequently, the relative configuration of
C(1), C(7a), and C(3a), was assigned as rel-(1S,7aR,3aR) (FIG. 3).

Example 5

Compound 1 Enhanced Adipocyte Differentiation in 3T3-L1 Cells

[0099] Preparation of 3T3-L1 Preadipocytes

[0100] Preadipocytes were prepared according to the method described by
Wald et al., (2007, "The small molecule harmine is an antidiabetic
cell-type-specific regulator of PPARγ expression." Cell Metabolism,
5(5): 357-370). Preadipocytes were cultured in DMEM (Dulbecco's Modified
Eagle's Medium-high glucose, Sigma D-7777) containing 10% of fetal bovine
serum (FBS) and incubated at 37° C. in a 5% CO2 incubator.

[0101] Before the differentiation induction experiments, the cells were
plated into either 96- or 24-well plates (the concentration of cells in
each well was about 2×104/cm2). The plates were incubated
for about two days to allow the cells to proliferate to confluence, and
then maintained for another two days. The medium was changed to different
differentiation media according to different experiments. The day of
switching to differentiation medium was designated as day zero.

[0102] Insulin Differentiation Model

[0103] The differentiation medium used in insulin differentiation model
was DMEM with 10% FBS comprising 10 μg/ml of insulin. Differentiation
media with various concentrations of Compound 1 were added to each well
of the 24- or 96-well 3T3-L1 preadipocyte plates (the final concentration
of Compound 1 in each well was 2 μg/ml, 5 μg/ml, 10 μg/ml, and
20 μg/ml, respectively). The control group was the culture with only
the differentiation medium and no sample. The cultures were incubated at
37° C. in a 5% CO2 incubator for seven days (during the
period, the differentiation medium and sample were replaced with fresh
ones twice), and then the medium was replaced by DMEM with 10% FBS. The
cultures were maintained until day 9 or day 10. The cells of the
experiment groups and control groups at day 9 or day 10 were stained with
AdipoRed and the concentrations of triglyceride were analyzed.

[0104] AdipoRed Staining

[0105] The 96-well plates were rinsed with PBS, and 200 μl of PBS and 5
μl of AdipoRed reagent (Lonze Walkersville, Inc., Walkersville, Md.,
USA, Catalog No. PT-7009) were added to each well. After 10 to 15
minutes, the plates were read with a spectrofluorometer (Infinite M200)
set at an excitation wavelength of 485 nm and an emission wavelength of
572 nm. The fluorescent data of the experiment group was divided by the
fluorescent data of the control group to obtain a percentage of induction
activity.

[0106] Measurement of Triglyceride Concentration

[0107] The 24-well plates were rinsed with PBS, and the cells were washed
off with 0.1 ml of 1% Triton X 100 per well. The cells were then frozen,
thawed and centrifuged (10,000 rpm/5 min) and the supernatant was
collected. An aliquot of 0.05 ml of the supernatant was analyzed using
Triglycerol assay Kit (Audit Diagnostics, Ltd.).

[0108] The protein amounts were analyzed using Bio-Rad Dc Protein assay
reagent (Bio-Rad). The triglyceride concentration was divided by the
protein concentration obtained using Bio-Rad Dc Protein assay to
calculate the amount (μg) of triglyceride per μg protein. The
triglyceride amounts of the experiment groups were divided by those of
the control groups to determine the differentiation induction activity of
the samples.

[0109] Results

[0110] FIGS. 4(A) to 4(D) show the effects of insulin and various
concentrations of Compound 1 on the differentiation of 3T3-L1
preadipocytes in insulin differentiation model observed under an inverted
microscope. The red color indicates the abundance of triglyceride
droplets present in differentiated adipocytes. It was found that the
cultures treated with 2 μg/ml (FIG. 4B), 5 μg/ml (FIG. 4C), and 10
μg/ml (FIG. 4D) Compound 1, respectively, contained more triglyceride
droplets than the control culture (FIG. 4A), and the quantity of
triglyceride droplets increased with dose of Compound 1 indicating that
Compound 1 can significantly promote the differentiation of 3T3-L1
preadipocytes.

[0111] The extent of enhancement of Compound 1 and troglitazone on 3T3-L1
differentiation were shown in Table 2.

[0112] It is found that Compound 1 can promote the differentiation of
3T3-L1 preadipocytes in insulin model.

Example 6

Compound 1 Demonstrating PPAR-65 Binding Activity

[0113] 10, 20, 40, 80 and 160 μg/ml of Compound 1 were tested in the
ligand binding assay conducted following the manuals described in
LanthaScreen TR-FRET PPAR Competitive Binding Assay kits (Invitrogen New
York, N.Y. Catalog no. PV4894). In a 96-well microtiter plate, the
reagents were pipetted into each well in the following order: 20 μL
2× Test Compound, 10 μL 4× Fluormone® Pan-PPAR Green
and 10 μL 4× PPARγ-LBD/Tb-anti-GST Ab in a total assay
volume of 40 μl/well. The reagents were gently mixed by placing the
microtiter plate on an orbital plate shaker for 30 seconds. The samples
were incubated at room temperature (20-25° C.) for 2 hours in the
dark allowing the binding of the samples to reach equilibrium. The
fluorescent emission signal of each well was measured at 495 nm and 520
nm with an Absorbance Microplate Reader (Spectra Max M5 from Molecular
Devices LLC).

[0114] The results show that Compound 1 can bind PPARγ with an
IC50 value of 68±21 μg/mL.

[0115] The upstream genes that regulate adipocyte differentiation have
been previously reported (F. M. Gregoire, C. M. Smas and H. S. Sul,
Understanding adipocyte differentiation. Physiological Reviews, 783
(1998), pp. 783-809.) Among them, aP2, LPL, PPARγ, adiponectin,
GLUT4, GLUT1, and C/EBPα were assayed to monitor the regulation of
3T3-L1 preadipocyte differentiation. The housekeeping genes of
β-actin and Glyceraldehyde 3-phosphate dehydrogenase (GADPH) were
used as control. "do" represents the conditions of the 3T3-L1
preadipocytes before adding inducing agents. In the "NC" groups, the
3T3-L1 preadipocytes were not treated with any inducing agent. Both "do"
and "NC" groups were the control groups. In the groups of "insulin," "E,"
and "troglitazone," the 3T3-L1 preadipocytes were induced with insulin,
Compound 1 and insulin, and troglitazone, respectively.

[0116] After culturing the 3T3-L1 cells for 9 days, insulin, Compound 1
and insulin, and troglitazone were added to the medium. The cells were
further cultivated, and the samples were collected on day 4 (early
adipocyte differentiation stage), day 7 (middle stage) and day 9 (late
stage) for the following assays.

[0117] RNA Preparation and Quantitative Real-Time PCR

[0118] Total RNA was extracted from 3T3-L1 cells using TRIZOL reagent
according to the manufacturer's instructions (GIBCO 15596-026; Molecular
Research Center, Inc). 4 μg of total RNA from each sample were
reverse-transcribed to cDNA according to the protocol of the reverse
transcript system [RevertAid H Minus First Strand cDNA Synthesis Kit
(Fementas K1632)] with the oligo (dT)18 primers. A TaqMan One-Step RT-PCR
Master Mix reagent kit and inventory primer and probes (Applied
Biosystems, Foster, Calif.) were applied for the quantitative real-time
PCR followed by a TaqMan gene expression assay. The sequences aP2
(Mm00445878_ml), LPL (Mm00434770_ml), PPARγ (Mm01184322_ml),
adiponectin (Mm00456425_ml), GLUT4 (Mm01245502_ml), GLUT1
(Mm01192270_ml), C/EBPα (Mm01265914_sl), C/EBPδ
(Mm00786711_sl), β-actin (Mm01205647_gl) and GADPH (Mm99999915_gl)
were amplified in the PCR. Levels of mRNAs for housekeeping genes
(β-actin and GADPH) were also measured by the real-time PCR. After
cDNA synthesis, the quantitative real-time PCR was performed with the ABI
Prism 7300 instrument (Applied Biosystems, Foster City, Calif., USA)
according to the manufacturer's protocol. The PCR conditions were 1 cycle
of 50 ° C. for 2 min, then 95 ° C. for 10 min, followed by
40 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 min.
The quantification was performed in duplicate and the experiments were
repeated independently three times.

[0119] FIG. 5 shows that the expressions of C/EBPα, PPARγ and
andadiponectin in the E group increase about 50% as compared to the
insulin group at the early adipocyte differentiation stage (day 4), but
the expression of GluT1 is 0.6 fold of the insulin group. FIG. 6 shows
that at the middle adipocyte differentiation stage (day 7), the
expression of PPARγ is elevated slightly (1.8 fold) in the E group,
but the expressions of aP2, LPL and GluT1 are dramatically lowered (0.3
fold) as compared to the insulin group. FIG. 7 shows that at the late
adipocyte differentiation stage (day 9), the expression of C/EBPα,
PPARγ and adiponectin in the E group is elevated slightly (1.59,
1.35, 1.49 folds, respectively) as compared to the insulin group, but the
expression of GluT1 is 0.6 fold of the insulin control.

[0120] The results show that Compound 1 can stimulate the expressions of
PPARγ and C/EBPα at the early, middle and late adipocyte
differentiation stages, but has little effect on the expression of aP2,
LPL, adiponectin, GLUT4, and GLUT1. This demonstrates that Compound 1
enhances the differentiation of preadipocytes to adipocytes through the
PPARγ/C/EBPα pathway (Evan D. Rosen, Chung-Hsin Hsu, Xinzhong
Wang, Shuichi Sakai, Mason W. Freeman, Frank J. Gonzalez, and Bruce M.
Spiegelman. (2002) C/EBPα induces adipogenesis through PPARγ:
a unified pathway. Genes Dev. 16(1): 22-26).

[0121] While the present invention has been described in conjunction with
the specific embodiments set forth above, many alternatives thereto and
modifications and variations thereof will be apparent to those of
ordinary skill in the art. All such alternatives, modifications and
variations are regarded as falling within the scope of the present
invention.

Patent applications by Gwo-Fang Yuan, Hsinchu City TW

Patent applications by Hsuen-Chun Liao, Hsinchu City TW

Patent applications by Kai-Ping Chen, Hsinchu City TW

Patent applications by Ming-Der Wu, Hsinchu City TW

Patent applications by Ming-Jen Cheng, Hsinchu City TW

Patent applications by Ping-Hsun Yang, Hsinchu City TW

Patent applications by Shie-Jea Lin, Hsinchu City TW

Patent applications by Yen-Lin Chen, Hsinchu City TW

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